Prion protein dimers useful for vaccination

ABSTRACT

Described is a prion protein, wherein said prion protein is a homodimer or heterodimer. The prion protein dimers are highly immunogenic capable of inducing an immune response in a mammal, thus useful for prophylactic or therapeutic vaccination against diseases associated with the infectious forms of prion proteins, PrP Sc , i.e. transmissible spongiform encephalopathies. Moreover, antibodies generated by using the prion protein diners as an antigen, pharmaceutical composition containing the prion protein dimers or antibodies directed against said diners as well as DNA sequences encoding the prion protein diners are described.

[0001] The present invention relates to a prion protein, wherein saidprion protein is a homodimer or heterodimer. The prion protein dimersare highly immunogenic capable of inducing an immune response in amammal, thus useful for prophylactic or therapeutic vaccination againstdiseases associated with the infectious forms of prion proteins,PrP^(Sc), i.e. transmissible spongiform encephalopathies. Furthermore,the present invention relates to antibodies generated by using the prionprotein dimers as an antigen, pharmaceutical compositions containing theprion protein dimers or antibodies directed against said dimers as wellas DNA sequences encoding the prion protein dimers.

[0002] Transmissible spongiform encephalopathies are neurodegenerativediseases such as scrapie of sheep, bovine spongiform encephalopathy(BSE) of cattle and Creutzfeldt-Jakob disease (CJD) of man. Infectiouspreparations derived from infected brains are resistant to ultravioletand ionizing radiation as well as other procedures which normallyinactivate nucleic acids indicating that nucleic acids are not requiredfor infectivity. Purification of infectious preparations from brainsrevealed the presence of a protein required for infectivity. Theseexperimental observations led to the “protein only” hypothesis whichproposes that particular proteinaceous infectious particles (prions) areresponsible for the transmission of transmissible spongiformencephalopathies (Prusiner et al., Proc. Natl. Acad. Sci. USA 95,13363-13383 (1998)). Prions consist mainly of a protease resistentprotein designated PrP^(Sc) (prion protein, “Sc”: scrapie), which is anabnormal isoform of the proteinase K sensitive PrP^(C) (“C”:cellular) .The prion protein PrP^(C) is a species specific protein which isphysiologically expressed in brain cells and peripheral blood cells aswell as, e.g., in spleen. The biological function of PrP^(C) is so farunknown. Transgenic mice which are no longer capable of expressingPrP^(C) (PrP knock out mice) do not show any pathological damages andcannot be infected with prions (Bueler et al., Nature 356, 577-582(1992); Bueler et al., Cell 73, 1339-1347 (1993)).

[0003] Both isoforms, PrP^(Sc) and PrP^(C), share the same amino acidsequence, but differ in their secondary structure. Circular Dichroism(CD) and Fourier Transform Infrared (FTIR) spectroscopy revealed asignificantly higher β-sheet content for PrP^(Sc) as compared to a highα-helix content in PrP^(C). It has been suggested that prion propagationinvolves the conversion of α-helical domains in PrP^(C) into β-sheets inPrP^(Sc). The in vitro conversion of PrP^(C) into a PrP^(Sc)-likemolecule was demonstrated employing a proteinase K resistance assay.

[0004] It has been suggested that for the development of a transmissiblespongiform encephalopathy the interaction of PrP^(Sc) and PrP^(C) is acrucial event, wherein PrP^(Sc) forces a conversion of PrP^(C) into thepathological conformation (PrP^(Sc)) Accordingly, transgenic mice whichare no longer capable of expressing PrP^(C) (PrP knock out mice) areresistant to infections with PrP^(Sc). Although, the prion proteins ofvarious species differ as regards their amino acid sequences, within thegroup of mammals there is a high homology (88 to 99%). Apparently, dueto differences in the amino acid sequences the prion protein cannot betransmitted between certain species (relative or absolute speciesbarrier). Due to the fact that the prion protein is a physiologicallyexpressed protein, each species exhibits immunological tolerance via itsown specific prion protein. Unfortunately, so far transmissiblespongiform encephalopathies, e.g. BSE, are incurable. There is notherapy available for soothing the symptoms of the disease, let alonefor stopping the disease, e.g. by vaccination.

[0005] Therefore, it is the object of the present invention to provide avaccine for the prevention or treatment of a transmissible spongiformencephalopathy.

[0006] According to the invention this is achieved by the subjectmatters defined in the claims.

[0007] The present invention provides a prion protein, wherein saidprion protein is a homodimer or heterodimer with the monomercorresponding to or comprising a prion protein according to thedefinition below. During the experiments leading to the presentinvention, it has, surprisingly, been found that prion protein dimersare useful for inducing an enhanced immune response against PrP^(c) andPrP^(Sc). Using this apparent remarkable immunogenicity of the prionprotein dimer it was even possible in vivo to generate antibodiesdirected against the homologous prion protein (i.e. mouse antibodiesdirected against murine PrP), thereby abolishing or by-passing the knownanto-tolerance present within a given species. After incubation of cellspermanently producing PrP^(Sc) with the antibodies that were generatedby immunization with the prion protein dimer for 16 hours a drasticinhibition of the de novo synthesis of PrP^(Sc) could be observed.Presumably, this result is due to generation of a functional PrP^(c)knock out state where the surface-located PrP^(c) substrate needed forcontinuous cellular conversion into PrP^(sc) is functionally impaired,resulting finally in inhibition of generation of PrP^(sc). It is knownfrom the literature that the surface location of PrP^(c) is aprerequisite for cellular prion conversion (Taraboulos et al., J. CellBiol. 129, 121-132 (1995)). Even more, studies with transgenic mice havedemonstrated that peripheral expression of PrP^(c) is absolutelyrequired for replication and transport of PrP^(sc) from peripheral sitesof the body to the central nervous system (Blättler et al., Nature 389,69-73 (1997)). In this respect it has been discovered by the inventorsthat after application of the prion protein dimer of the presentapplication to a subject, the induced immune response results in thedownregulation of the surface expression of PrP^(c). Accordingly, theconversion rate (PrP^(c)

PrP^(Sc)) can be drastically reduced, thus, blocking the initiation andthe progression of the disease. Thus, the prion protein dimers of thepresent invention are useful as a vaccine for the prevention ortreatment of a transmissible spongiform encephalopathy.

[0008] Accordingly, the present invention relates to a prion protein,wherein said prion protein is a homodimer or heterodimer which canelicit an immune response against PrP^(C) or PrP^(Sc), preferably animmune response which is higher compared to the immune response obtainedusing for vaccination the corresponding monomer. As used herein, theterm “prion protein” comprises the native (full length and/or mature)protein as well as variants thereof, e.g. truncated versions, which arestill useful for eliciting an enhanced immune response, i.e. stilluseful for vaccination. In this context, the term “protein” alsocomprises peptides or polypeptides having a length of at least 8,preferably of at least 15 and, more preferably, of at least 20 aminoacids. The person skilled in the art knows sources for the prion proteinmonomers and DNA sequences encoding the prion protein monomers,respectively (Schätzl et al., J. Mol. Biol. 245, 362-374 (1995); Wopfneret al., J. Mol. Biol. 289, 1163-1178 (1999); sequences deposited atGenbank accessible via Public Medline database). Furthermore, a personskilled in the art knows methods for coupling the monomers in order toobtain the dimer. Preferably, the monomers are covalently coupled.

[0009] In a preferred embodiment, the homodimer or heterodimer is afusion protein comprising the monomeric prion protein units. Such afusion protein can be generated by methods well known by the personskilled in the art. The selection of a particular method mainly dependson the length of the monomers, i.e. very short fusion proteins arepreferably chemically synthesized, e.g. by standard solid phasesynthesis, whereas longer fusion proteins are preferably produced byexpression of the corresponding DNA sequences in a suitable host andisolation/purification of the fusion protein from the host or theculture medium.

[0010] Generally, by means of conventional molecular biologicalprocesses (see, e.g., Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2^(nd) edition Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) it is not only possible to generate a DNAsequence encoding the desired fusion protein but, additionally, tointroduce different mutations into the DNA sequence. One possibility isthe production of deletion mutants in which DNA molecules are producedby continuous deletions from the 5′- or 3′-termini of the coding DNAsequence and that lead to the synthesis of proteins that are shortenedaccordingly. The person skilled in the art can easily check as towhether such prion protein diners containing shortened monomeric unitsare still useful as a vaccine, e.g. using the methods described in theexamples, below. For the manipulation in prokaryotic cells by means ofgenetic engineering the DNA sequences can be introduced into plasmidsallowing a mutagenesis or a modification of a sequence by recombinationof DNA sequences. By means of conventional methods (cf. Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, NY, USA) bases can be exchanged andnatural or synthetic sequences can be added. Furthermore, manipulationscan be performed that provide suitable cleavage sites or that removesuperfluous DNA or cleavage sites. If insertions, deletions orsubstitutions are possible, in vitro mutagenesis, primer repair,restriction or ligation can be performed. As analysis method usuallysequence analysis, restriction analysis and other biochemical ormolecular biological methods are used.

[0011] In a more preferred embodiment, the monomers of the fusionprotein are linked via a peptide spacer. Suitable peptide spacers, whichpreferably consist of non-polar and non-bulky amino acids, have a lengthbelow 20 residues, provide a certain flexibility, thereby avoiding e.g.cystein or prolin residues which can induce rigidity (or vice versa incases when rigidity or induction of an orientation/turns are wanted).These spacers are known to the person skilled in the art and comprisefor example AGAIGGA [HIV-1 protease; Kräusslich et al., Proc. Natl.Acad. Sci. USA, 88, 3212-3217 (1991)], SGGRGG [HIV gp41; Tan et al.,Proc. Natl. Acad. Sci USA, 94, 12303-12308 (1991)], GSGGGGSGGGGSGGSGA[single chain T-cell receptor; Khandekar et al., J. Biol. Chem. 272,32190-32197 (1997)] or (GGGGS)_(n=1-3) [single chain antibodies; Korttet al., Eur. J. Biochem. 221, 151-157 (1994)]. Examples of preferredspacers are (a) spacers having a length of 2 to 3 amino acids, whereinthe DNA sequence encoding said spacer contains a restriction site, (b)oligomers composed of small neutral amino acids (e.g.Ala-Gly-Ala-Ile-Gly-Gly-Ala), or c) an oligomer having an amino acidsequence that defines an epitope, e.g. flag-tag, HA-epitope etc..

[0012] For the generation of the prion protein dimers encoding DNAsequences and for constructing expression vectors which contain said DNAsequences, it is possible to use general methods known in the art. Thesemethods include e.g. in vitro recombination techniques, syntheticmethods and in vivo recombination methods as described in Sambrook etal., Molecular Cloning, A Laboratory Manual, 2^(nd) edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., for example.

[0013] For recombinant production of the dimeric prion protein of theinvention, the DNA sequences encoding said protein are inserted into asuitable expression vector. Preferably, they are plasmids, cosmids,viruses, bacteriophages and other expression vectors usually used in thefield of genetic engineering. Expression vectors suitable for use in thepresent invention include, but are not limited to e.g. pGEMEX, pUCderivatives, pGEX-2T, pET3b, T7 based expression vectors and pQE andpTrcHis (for expression in E.coli). For the expression in yeast, e.g.pY100 and Ycpad1 have to be mentioned while e.g. pcDNA3.1, pKCR, pEFBOS,cDM8, pMSCND, and pCEV4 have to be indicated for the expression inanimal cells. The baculovirus expression vector pAcSGHisNT-A isespecially suitable for the expression in insect cells. The DNAsequences can also be contained in a recombinant virus containingappropriate expression cassettes. Suitable viruses that may be used inthe present invention include baculovirus, SFV, vaccinia, sindbis virus,SV40, Sendai virus, adenovirus, an AAV virus or a parvovirus, such asMVM or H-1. The vector may also be a retrovirus, such as MOMULV, MoMuLV,HaMuSV, MuMTV, RSV or GaLV. Preferably, the DNA sequence is operativelylinked to the regulatory elements in the recombinant vector thatguarantee the transcription and synthesis of an RNA in prokaryoticand/or eukaryotic cells that can be translated. The nucleotide sequenceto be transcribed can be operably linked to a promoter like a T7,metallothionein I or polyhedrin promoter. For expression in mammals, theDNA sequences are operatively linked to a suitable promoter like, e.g. ahuman cytomegalovirus “immediate early promoter” (pCMV).

[0014] Suitable host cells include bacteria, yeast, insect and animalcells, preferably mammalian cells. The E. coli strains HB101, DH1,x1776, JM101, JM109, BL21, XL1Blue and SG 13009, the yeast strainSaccharomyces cerevisiae, the insect cells sf9 and the animal cells L,A9, 3T3, FM3A, BHK, human SW13, CHO, COS, Vero and HeLa are preferred.Methods of transforming these host cells, of phenotypically selectingtransformants and of expressing the DNA according to the invention byusing the above described vectors are known in the art. For recombinantproduction of the dimeric prion protein of the invention, the host cellis cultivated under conditions allowing the synthesis of the protein andthe protein is subsequently isolated from the cultivated cells and/orthe culture medium. Isolation and purification of the recombinantlyproduced proteins may be carried out by conventional means includingpreparative chromatography and immunological separations involvingaffinity chromatography with monoclonal or polyclonal antibodies, e.g.with the antibody A7 and 3F4 described in the examples, below.

[0015] In an alternative preferred embodiment the monomers of thedimeric prion protein of the invention are covalently coupled viasuitable compounds, e.g. in such a way that the monomers are covalentlylinked via their C-termini or N-termini or via the C-terminus of onemonomer and the N-terminus of the other monomer. Care has to be takenthat dimerization is carried out in such a way that eliciting an immuneresponse by the dimeric protein is not adversely effected. Suitablecompounds for dimerization are known to the person skilled in the art,preferably such a compound is a polyethylenglycole, activatedbenzodiazepine, oxazolone, aminimide, azalactone, diketopiperazine or amonosaccharide. The corresponding chemical reactions are also known tothe person skilled in the art.

[0016] The monomers of the dimeric prion protein of the presentinvention can be identical (homodimer: the monomers are derived from thesame species) or different (heterodimer: the monomers are derived fromdifferent species). In the case of the heterodimer it can be expectedthat an immune response against the own PrP^(C) as well as the foreignPrP^(C) is induced.

[0017] As already described above the dimeric prion protein of thepresent invention also includes monomers which are variants of thenative prion protein monomers. Particularly preferred are variants whichare N-terminally and/or C-terminally truncated proteins (monomers).

[0018] A preferred dimeric prion protein (and its correspondingnucleotide sequence) is shown in FIG. 5.

[0019] The present invention also relates to the use of a dimeric prionprotein of the present invention for the production of polyclonal ormonoclonal antibodies as well as to an antibody, preferably a monoclonalantibody which binds to the dimeric prion protein of the presentinvention. Such antibodies are useful for passive immunization and canbe produced according to well known methods. The term “antibody”,preferably, relates to antibodies which consist essentially of pooledmonoclonal antibodies with different epitopic specificities, as well asdistinct monoclonal antibody preparations. Monoclonal antibodies aremade using as an antigen the dimeric prion protein of the invention bymethods well known to those skilled in the art (see, e.g., Köhler etal., Nature 256 (1975), 495). As used herein, the term “antibody” (Ab)or “monoclonal antibody” (Mab) is meant to include intact molecules aswell as antibody fragments (such as, for example, Fab and F(ab′)2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation, and may have less non-specific tissuebinding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325(1983).) Thus, these fragments are preferred, as well as the products ofa FAB or other immunoglobulin expression library. Moreover, antibodiesof the present invention include chimeric, single chain (scFv), andhumanized antibodies.

[0020] Moreover, the present invention also relates to DNA sequencesencoding the dimeric prion proteins of the present invention as well asan expression vector containing such a DNA sequence. Examples of suchDNA sequences and suitable expression vectors are already describedabove. Such DNA sequences or expression vectors are not only useful forthe recombinant production of the dimeric prion protein of the presentinvention but also useful as “genetic vaccine”. For the preparation of agenetic vaccine, the DNA sequences of the invention are inserted into anappropriate vector under the control of a suitable promoter, e.g. avector or promoter as described above. Additional suitable vectors are,e.g., herpes simplex virus type 1 amplicon or recombinant Semliki forestvirus vectors, plus-strand RNA virus vectors (lentiviral vectors),minus-strand RNA virus vectors (e.g. Rabies virus, RSV) . In thisconnection also “gene gun” applications are suitable. A vaccineaccording to the present invention can be used to stimulate humoraland/or cellular immune response in subjects who may benefit from suchresponses by protection against or treatment of a transmissiblespongiform encephalopathy.

[0021] Finally, the present invention relates to a pharmaceuticalcomposition comprising a dimeric prion protein, an antibody, DNAsequence or an expression vector of the present invention in apharmaceutically acceptable carrier. After application to the subject,the immune response induced should result in the downregulation of thesurface expression of PrP^(C). Accordingly, the transport of infectiousprions from peripheral sites of inoculation (e.g. oral or i.v.) to thecentral nervous system as well as the initial peripheral replicationshould be significantly impaired. In addition, the conversion rate(PrP^(C)

PrP^(Sc)) can be drastically reduced, thus blocking the progression ofthe disease. Thus, the compounds of the present invention are useful forthe preparation of a vaccine for the prevention or treatment of atransmissible spongiform encephalopathy (prophylactic or therapeuticvaccination). For the preparation of the vaccine the purified compoundsdescribed above may be combined with any suitable adjuvant such asISCOMS, alum, Freund's Incomplete or Complete Adjuvant, Quil A and othersaponins or any other adjuvant as described in the literature. Suitabledoses and routes of administration depend on the particular compound andare well known to the person skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1: PrP dimer

[0023] Coomassie blue staining after purification showing a purityof >90%. M, molecular weight standards.

[0024]FIG. 2: Characterization of the PrP dimer using CD

[0025] The results show that the PrP monomer and the PrP dimer have asimilar or even identical secondary structure.

[0026]FIG. 3

[0027] (a) Immunological responses in different mice

[0028] Immunization with the PrP monomer and PrP dimer, respectively.Recombinant murine PrP was separated on a SDS PAGE gel, subsequentlytransferred to a PVDF membrane and the immunoblots were probed withdecreasing concentrations auf mice antisera. The immunization with thePrP dimer resulted in an enhanced antibody production.

[0029] (b) Immunological responses in different rabbits

[0030] Immunization with the PrP monomer and PrP dimer, respectively.Recombinant PrP monomer (PrP_(M)) and PrP dimer (PrP_(D)), respectively,were separated on a SDS PAGE gel, subsequently transferred to a PVDFmembrane and the immunoblots were probed with anti-PrP_(monomer) andanti-PrP_(dimer), respectively. The immunization with the PrP dimerresulted in an enhanced antibody production.

[0031]FIG. 4: In vitro experiments using PrP^(Sc) infected cells

[0032] During incubation with antibodies for 16 hours proteins weremetabolically labeled using ³⁵S-methionine and subsequentlyimmunoprecipitated (RIPA). Prior to the RIPA the cell lysates wereseparated into two fractions for digestion with proteinase K (+/−) andseparated into soluble and insoluble fractions using ultracentrifugation(1 hour, 100,000×g, 1% sarcosyl); data only shown for the insolublefraction. Only after incubation with A7 a drastic decrease of the denovo synthesis of PrP^(Sc) can be observed. A,B: after deglycosylationusing PNGase F; K: without incubation with antibodies; P: preimmuneserum; 3F4: monoclonal antibody directed against the 3F4 epitope of theprion protein; A7: antibody obtained from rabbits which had beenimmunized with the PrP dimer.

[0033]FIG. 5: Translation of hisPrPDPcr (1-1320)

[0034] Underlined in the first line: His-Tag

[0035] Underlined in the middle: Linker

[0036] The present invention is explained by the examples.

EXAMPLE 1 Generation of a Fusion Protein (Dimer) Comprising Two PrionProtein Monomers Covalently Linked by a Peptide Linker

[0037] Mouse genomic DNA encoding the prion protein (murine PrP-A,derived from murine N2a neuroblastoma cell line; for sequence seeSchätzl et al, 1995, supra; Wopfner et al., 1999, supra) was amplifiedby polymerase chain reaction (PCR) using the following primer pairswhich introduce a peptide linker (Ala-Gly-Ala-Ile-Gly-Gly-Ala) encodingsequence containing a PvuI restriction site; primer pair I: 5′-GCG GATCCG TCG CCA CCA TGG CGA ACC TTG GCT A-3′ (PrPB+1) and 5′-ACC GAT CGC TCCAGC GCT GGA TCT TCT CCC GTC GTA AT-3′ (231+L new); primer pair II:5′-AGC GAT CGG TGG AGC TAA AAA GCG GCC AAA GCC TGG AG-3′ (23+L new);5′-ATC TAG ATC ATC ATC ATC CCA CGA TCA GGA AGA-3′ (254 PX) (the DNAsequence encoding the PvuI restriction site is underlined). For theintroduction of an N-terminal His-tag, the removal of the N-terminal andC-terminal signal peptides and the introduction of additionalrestriction sites, a second PCR was carried out using the followingprimers: 5′-GAT GTT GGA TCC TGC AAG AAG CGG CCA AAG-3′ (5′-primer, theBamHI-site is underlined.); 5′-GGA GGA GAT CCA GCA GCT AGA AGC TTT TC-3′(3′-primer, the HindIII-site is underlined.) The PCR fragment obtained(c.f. FIG. 5) was inserted into the pQE30 expression vector (Qiagen,Hilden, Del.). In order to obtain higher yields of the recombinantlyproduced protein, proteinase deficient bacteria (BL21, StratageneEurope, Amsterdam, The Netherlands) were used for expression. Four hoursafter induction of expression using 2 mM IPTG, bacteria were lysed in 6M guanidine hydrochloride (6 M guanidine hydrochloride, 20 mM sodiumphosphate, 500 mM sodium chloride, pH 7,8). cell debris were pelleted bycentrifugation (20 min., 10,000×g), the supernatant was loaded on aNi²⁺loaded SP-HiTrap column (Amersham Pharmacia, Uppsala, Sweden) whichhad been previously equilibrated using binding buffer (8 M urea, 20 mMsodium phosphate, 500 mM sodium chloride, pH 7.8) and incubated. Thecolumn was washed for several times (8 M urea, 20 mM sodium phosphate,500 mM sodium chloride, 80 mM imidazole, pH 6.3) and, subsequently, thefusion protein containing the His-tag was eluted using 8 M urea, 20 mMsodium phosphate, 500 mM sodium chloride, 500 mM imidazole, pH 6.3.Fractions of the eluate were collected. The fractions showing thehighest protein concentration were pooled and for removal of ureadialyzed against pure water (Slide-A-Lyzer, Pierce, Bonn, Del.). Asshown in FIG. 1, the protein could be obtained with a very high purity.As shown in FIG. 2, the secondary structure of the purified renatureddimer is similar (or even identical) to the secondary structure of theprion protein monomer.

EXAMPLE 2 Immunization of Mice and Rabbits

[0038] Ten female B6D₂F₁ (B57BL/6×DBA) mice (inbred strain generated atthe Institute of Molecular Animal Breeding/Gene Center Munich, Del.; sixweeks old) were immunized by subcutaneous injections of 50 μg of theprotein dimer described in Example 1 and a control monomer, respectively(monomer: murine PrP23-231 verus dimer: murine PrP23-231=23-231[connected by the linker sequence], both with the 3F4 epitope and anN-terminal poly-histidine tag). At day 0 the proteins were combined withFreund's Complete Adjuvant, for boostering (at day 21 and day 42,respectively) the proteins were combined with Freund's IncompleteAdjuvant. Ten days after the last immunization blood samples forantibody evaluation were taken. It could be shown that the applicationof the prion protein monomer (control) resulted in the generation of alower amount of specific antibodies compared to the application of theprion protein diner. After application of the dimer about 50% of miceshowed a pronounced accumulation of specific antibodies (FIG. 3a). Theinduced specific polyclonal immune response was measured in immunoblot.Recombinant murine PrP23-231 was separated on SDS-PAGE, transferred toPVDF membranes and incubated in an immunoblot analysis with serialdilutions of sera of immunized mice (primary antibody). The pre-immunesera were used to eliminate background signals. Staining wasaccomplished using conjugated secondary antibodies (anti-mouse) and theenhanced chemiluminescence kit (ECL plus, Amersham Corp.,Buckinghamshire, U.K.). The mice immunized with PrP dimer showed higherantibody titers. Dilutions beyond 1:3000 still resulted in detectablesignals. This finding indicates that the used immunogen was capable ofovercoming the auto-tolerance phenomenon known for species-identicalprion proteins. Further data showed that the induced antibodies were notdirected against the N-terminal fusion domain or the linker sequencebeing reactive against authentic murine PrP23-231.

[0039] A similar result could already be achieved using rabbits, i.e.the rabbits immunized with the prion protein dimer showed a strongerimmune response compared to the rabbits immunized with the prion proteinmonomer (FIG. 3b). Recombinant monomeric and dimeric prion proteins(PrPM and PrPD, respectively, which were used also as immunogens inimmunization) were separated on SDS-PAGE, transferred to PVDF membranesand analyzed in immunoblot. The left panel shows incubation with theserum from the rabbit immunized with PrP monomer, the right panel fromthe arbbit immunized with PrP dimer. Staining was accomplished usingconjugated anti-rabbit secondary antibodies and the ECL plus kit.Whereas the left panel shows mainly a reaction against the monomericPrP, the right panel demonstrates a much stronger immune reactivitywhich is directed against monomeric and dimeric prion protein.

EXAMPLE 3 The Application of Antibodies Generated by Immunization with aprion protein dimer Leads to Inhibition of the de novo Synthesis ofPrP^(Sc) in vitro

[0040] The biological relevance of the immunization experimentsdescribed in Example 2 could be verified by the following in vitroexperiment. ScMHM2 cells [cultured murine neuroblastoma cells (ATCC CCL131); persistently infected with RML prions; stably transfected with a3F4-tagged PrP; c.f. Schätzl et al., J. Virol. 71, 8821-8831, (1997)]which are infected with the infectious version of the prion protein(PrP^(Sc)) and permanently produce PrP^(Sc) were incubated withdifferent types of antibodies. Incubation was carried using (a)commercially available monoclonal antibodies (3F4) (Signet PathologySystems, Dedham, Mass., USA) which are directed against an epitope ofthe prion protein produced in ScMHM2 cells and (b) the antiserumobtained from rabbits immunized with the prion protein dimer describedin Example 1. During incubation with antibodies for 16 hours proteinswere metabolically labeled using ³⁵S-methionine and subsequentlyimmunoprecipitated (RIPA). Prior to the RIPA the cell lysates wereseparated into two fractions for digestion with proteinase K (+/−) andseparated into soluble and insoluble fractions using ultracentrifugation(1 hour, 100,000×g, 1% sarcosyl). As shown in FIG. 4, after incubationwith the prion protein dimer for 16 hours a drastic inhibition of the denovo synthesis of PrP^(Sc) could be observed. The results observed withthe 3FA antibody are less pronounced. It can be concluded that thisresult is due to generation of a functional PrP^(c) knock out state onthe cell surface and, accordingly, an inhibition of the generation ofPrP^(Sc). These results demonstrate in living cells that the inducedanti-PrP antibodies are capable of binding to surface-located authenticPrP^(c) under native conditions (proof of principle for interaction withsurface-located PrP under native conditions). Even more, boundantibodies are apparently internalised with PrP^(c) demonstrating a highbinding affinity (resulting in an insoluble PrP of full length which isPK sensitive, see full-length signal in FIG. 4A, lanes 5-8). Finally,endogeneous PrP^(Sc) biogenesis was heavily impaired resulting in nomore de novo generated PK resistant PrP^(Sc) (FIG. 4B, lanes 7 and 8).The biological effect on PrP^(Sc) biogenesis is therefore demonstratedas well (proof of principle for impairment of prion biogenesis). Theefficiency of this principle could also be shown by using particularchemical compounds: The functional removal of surface-PrP^(c) resultedin a reduction of the production of prion proteins.

1 15 1 1320 DNA Artificial Sequence Description of Artificial Sequencefusion protein 1 atg aga gga tcg cat cac cat cac cat cac gga tcc tgc aagaag cgg 48 Met Arg Gly Ser His His His His His His Gly Ser Cys Lys LysArg 1 5 10 15 cca aag cct gga ggg tgg aac act ggc gga agc cga tac cctggg cag 96 Pro Lys Pro Gly Gly Trp Asn Thr Gly Gly Ser Arg Tyr Pro GlyGln 20 25 30 ggg agc cct gga ggc aac cgt tac cca cct cag ggt ggc acc tggggg 144 Gly Ser Pro Gly Gly Asn Arg Tyr Pro Pro Gln Gly Gly Thr Trp Gly35 40 45 cag ccc cac ggt ggt ggc tgg gga caa ccc cat ggg ggc agc tgg gga192 Gln Pro His Gly Gly Gly Trp Gly Gln Pro His Gly Gly Ser Trp Gly 5055 60 caa cct cat ggt ggt agt tgg ggt cag ccc cat ggc ggt gga tgg ggc240 Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Gly Trp Gly 6570 75 80 caa gga ggg ggt acc cac aat cag tgg aac aag ccc agt aag cca aaa288 Gln Gly Gly Gly Thr His Asn Gln Trp Asn Lys Pro Ser Lys Pro Lys 8590 95 acc aac atg aag cac atg gcc ggc gct gct gcg gca ggg gcc gtg gtg336 Thr Asn Met Lys His Met Ala Gly Ala Ala Ala Ala Gly Ala Val Val 100105 110 ggg ggc ctt ggt ggc tac atg ctg ggg agt gcc atg agc agg ccc atg384 Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Met 115120 125 atc cat ttt ggc aac gac tgg gag gac cgc tac tac cgt gaa aac atg432 Ile His Phe Gly Asn Asp Trp Glu Asp Arg Tyr Tyr Arg Glu Asn Met 130135 140 tac cgc tac cct aac caa gtg tac tac agg cca gtg gat cag tac agc480 Tyr Arg Tyr Pro Asn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser 145150 155 160 aac cag aac aac ttc gtg cac gac tgc gtc aat atc acc atc aagcag 528 Asn Gln Asn Asn Phe Val His Asp Cys Val Asn Ile Thr Ile Lys Gln165 170 175 cac acg gtc acc acc acc acc aag ggg gag aac ttc acc gag accgat 576 His Thr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp180 185 190 gtg aag atg atg gag cgc gtg gtg gag cag atg tgc gtc acc cagtac 624 Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys Val Thr Gln Tyr195 200 205 cag aag gag tcc cag gcc tat tac gac ggg aga aga tcc agc gctgga 672 Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg Ser Ser Ala Gly210 215 220 gcg atc ggt gga gct aaa aag cgg cca aag cct gga ggg tgg aacact 720 Ala Ile Gly Gly Ala Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn Thr225 230 235 240 ggc gga agc cga tac cct ggg cag ggc agc cct gga ggc aaccgt tac 768 Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly Asn ArgTyr 245 250 255 cca cct cag ggt ggc acc tgg ggg cag ccc cac ggt ggt ggctgg gga 816 Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly Gly Gly TrpGly 260 265 270 caa ccc cat ggg ggc agc tgg gga caa cct cat ggt ggt agttgg ggt 864 Gln Pro His Gly Gly Ser Trp Gly Gln Pro His Gly Gly Ser TrpGly 275 280 285 cag ccc cat ggc ggt gga tgg ggc caa gga ggg ggt acc cacaat cag 912 Gln Pro His Gly Gly Gly Trp Gly Gln Gly Gly Gly Thr His AsnGln 290 295 300 tgg aac aag ccc aat aag cca aaa acc aac atg aag cac atggcc ggc 960 Trp Asn Lys Pro Asn Lys Pro Lys Thr Asn Met Lys His Met AlaGly 305 310 315 320 gct gct gcg gca ggg gcc gtg gtg ggg ggc ctt ggt ggctac atg ctg 1008 Ala Ala Ala Ala Gly Ala Val Val Gly Gly Leu Gly Gly TyrMet Leu 325 330 335 ggg agc gcc atg agc agg ccc atg atc cat ttt ggc aacgac tgg gag 1056 Gly Ser Ala Met Ser Arg Pro Met Ile His Phe Gly Asn AspTrp Glu 340 345 350 gac cgc tac tac cgt gaa aac atg tac cgc tac cct aaccaa gtg tac 1104 Asp Arg Tyr Tyr Arg Glu Asn Met Tyr Arg Tyr Pro Asn GlnVal Tyr 355 360 365 tac agg cca gtg gat cag tac agc aac cag aac aac ttcgtg cac gac 1152 Tyr Arg Pro Val Asp Gln Tyr Ser Asn Gln Asn Asn Phe ValHis Asp 370 375 380 tgc gtc aat atc acc atc aag cag cac acg gtc acc accacc acc aag 1200 Cys Val Asn Ile Thr Ile Lys Gln His Thr Val Thr Thr ThrThr Lys 385 390 395 400 ggg gag aac ttc acc gag acc gat gtg aag atg atggag cgc gtg gtg 1248 Gly Glu Asn Phe Thr Glu Thr Asp Val Lys Met Met GluArg Val Val 405 410 415 gag cag atg tgc gtc acc cag tac cag aag gag tcccag gcc tat tac 1296 Glu Gln Met Cys Val Thr Gln Tyr Gln Lys Glu Ser GlnAla Tyr Tyr 420 425 430 gac ggg aga aga tcc agc agc tag 1320 Asp Gly ArgArg Ser Ser Ser 435 2 439 PRT Artificial Sequence Description ofArtificial Sequence fusion protein 2 Met Arg Gly Ser His His His His HisHis Gly Ser Cys Lys Lys Arg 1 5 10 15 Pro Lys Pro Gly Gly Trp Asn ThrGly Gly Ser Arg Tyr Pro Gly Gln 20 25 30 Gly Ser Pro Gly Gly Asn Arg TyrPro Pro Gln Gly Gly Thr Trp Gly 35 40 45 Gln Pro His Gly Gly Gly Trp GlyGln Pro His Gly Gly Ser Trp Gly 50 55 60 Gln Pro His Gly Gly Ser Trp GlyGln Pro His Gly Gly Gly Trp Gly 65 70 75 80 Gln Gly Gly Gly Thr His AsnGln Trp Asn Lys Pro Ser Lys Pro Lys 85 90 95 Thr Asn Met Lys His Met AlaGly Ala Ala Ala Ala Gly Ala Val Val 100 105 110 Gly Gly Leu Gly Gly TyrMet Leu Gly Ser Ala Met Ser Arg Pro Met 115 120 125 Ile His Phe Gly AsnAsp Trp Glu Asp Arg Tyr Tyr Arg Glu Asn Met 130 135 140 Tyr Arg Tyr ProAsn Gln Val Tyr Tyr Arg Pro Val Asp Gln Tyr Ser 145 150 155 160 Asn GlnAsn Asn Phe Val His Asp Cys Val Asn Ile Thr Ile Lys Gln 165 170 175 HisThr Val Thr Thr Thr Thr Lys Gly Glu Asn Phe Thr Glu Thr Asp 180 185 190Val Lys Met Met Glu Arg Val Val Glu Gln Met Cys Val Thr Gln Tyr 195 200205 Gln Lys Glu Ser Gln Ala Tyr Tyr Asp Gly Arg Arg Ser Ser Ala Gly 210215 220 Ala Ile Gly Gly Ala Lys Lys Arg Pro Lys Pro Gly Gly Trp Asn Thr225 230 235 240 Gly Gly Ser Arg Tyr Pro Gly Gln Gly Ser Pro Gly Gly AsnArg Tyr 245 250 255 Pro Pro Gln Gly Gly Thr Trp Gly Gln Pro His Gly GlyGly Trp Gly 260 265 270 Gln Pro His Gly Gly Ser Trp Gly Gln Pro His GlyGly Ser Trp Gly 275 280 285 Gln Pro His Gly Gly Gly Trp Gly Gln Gly GlyGly Thr His Asn Gln 290 295 300 Trp Asn Lys Pro Asn Lys Pro Lys Thr AsnMet Lys His Met Ala Gly 305 310 315 320 Ala Ala Ala Ala Gly Ala Val ValGly Gly Leu Gly Gly Tyr Met Leu 325 330 335 Gly Ser Ala Met Ser Arg ProMet Ile His Phe Gly Asn Asp Trp Glu 340 345 350 Asp Arg Tyr Tyr Arg GluAsn Met Tyr Arg Tyr Pro Asn Gln Val Tyr 355 360 365 Tyr Arg Pro Val AspGln Tyr Ser Asn Gln Asn Asn Phe Val His Asp 370 375 380 Cys Val Asn IleThr Ile Lys Gln His Thr Val Thr Thr Thr Thr Lys 385 390 395 400 Gly GluAsn Phe Thr Glu Thr Asp Val Lys Met Met Glu Arg Val Val 405 410 415 GluGln Met Cys Val Thr Gln Tyr Gln Lys Glu Ser Gln Ala Tyr Tyr 420 425 430Asp Gly Arg Arg Ser Ser Ser 435 3 7 PRT Artificial Sequence Descriptionof Artificial Sequence peptide spacer 3 Ala Gly Ala Ile Gly Gly Ala 5 46 PRT Artificial Sequence Description of Artificial Sequence peptidespacer 4 Ser Gly Gly Arg Gly Gly 5 5 17 PRT Artificial SequenceDescription of Artificial Sequence peptide spacer 5 Gly Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly 5 10 15 Ala 6 5 PRTArtificial Sequence Description of Artificial Sequence peptide spacer 6Gly Gly Gly Gly Ser 5 7 10 PRT Artificial Sequence Description ofArtificial Sequence peptide spacer 7 Gly Gly Gly Gly Ser Gly Gly Gly GlySer 5 10 8 15 PRT Artificial Sequence Description of Artificial Sequencepeptide spacer 8 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly GlySer 5 10 15 9 7 PRT Artificial Sequence Description of ArtificialSequence peptide spacer 9 Ala Gly Ala Ile Gly Gly Ala 5 10 34 DNAArtificial Sequence Description of Artificial Sequence Primer 10gcggatccgt cgccaccatg gcgaaccttg gcta 34 11 38 DNA Artificial SequenceDescription of Artificial Sequence Primer 11 accgatcgct ccagcgctggatcttctccc gtcgtaat 38 12 38 DNA Artificial Sequence Description ofArtificial Sequence Primer 12 agcgatcggt ggagctaaaa agcggccaaa gcctggag38 13 33 DNA Artificial Sequence Description of Artificial SequencePrimer 13 atctagatca tcatcatccc acgatcagga aga 33 14 30 DNA ArtificialSequence Description of Artificial Sequence Primer 14 gatgttggatcctgcaagaa gcggccaaag 30 15 29 DNA Artificial Sequence Description ofArtificial Sequence Primer 15 ggaggagatc cagcagctag aagcttttc 29

1. A prion protein, wherein said prion protein is a homodimer or heterodimer.
 2. The prion protein of claim 1, wherein the monomers of the homodimer or heterodimer are covalently coupled.
 3. The prion protein of claim 2, wherein the homodimer or heterodimer is a fusion protein comprising the monomers.
 4. The prion protein of claim 3, wherein the monomers of the fusion protein are linked via a peptide spacer.
 5. The prion protein of claim 4, wherein the peptide spacer is (a) a spacer having a length of 2 to 3 amino acids, wherein the DNA sequence encoding said spacer contains a restriction site or (b) an oligomer composed of small neutral amino acids or c) an oligomer having an amino acid sequence that defines an epitope.
 6. The prion protein of claim 1 or 2, wherein the monomers are covalently coupled via polyethylenglycole, activated benzodiazepine, oxazolone, azalactone, diketopiperazine or a monosaccharide.
 7. The prion protein of any one of claims 1 to 6, wherein the monomers are derived from the same or different species.
 8. The prion protein of any one of claims 1 to 7, wherein the monomers comprise the native prion protein or a variant thereof.
 9. The prion protein of claim 8, wherein the variant is a N-terminally and/or C-terminally truncated prion protein.
 10. Use of a prion protein according to any one of claims 1 to 9 for the production of a polyclonal or monoclonal antibody.
 11. An antibody which specifically binds to a prion protein according to any one of claims 1 to
 9. 12. The antibody of claim 11 which is a monoclonal antibody.
 13. A DNA sequence encoding the prion protein of any one of claims 1 to
 9. 14. An expression vector containing a DNA sequence of claims
 13. 15. A pharmaceutical composition containing a prion protein according to any one of claims 1 to 9, an antibody of claim 11 or 12, a DNA sequence of claim 13 or an expression vector of claim
 14. 16. Use of a prion protein according to any one of claims 1 to 9, an antibody of claim 11 or 12, a DNA sequence of claim 13 or an expression vector of claim 14 for the preparation of a vaccine for the prevention or treatment of a transmissible spongiform encephalopathy. 